Streptococcus suis 5’-nucleotidases contribute to adenosine-mediated immune evasion and virulence in a mouse model

ABSTRACT Streptococcus suis (S. suis) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5’-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5’-nucleotidases (Snts) within S. suis, named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His–Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) via the Ado-A2aR pathway in vitro, and within murine blood and organs in vivo. This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, S. suis 5’-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs in vivo. This elucidation of their integral functions in the pathogenic process of S. suis not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing S. suis infections.


Introduction
The innate immune system is a highly regulated and interconnected cellular network that collaboratively maintains and restores host homoeostasis while protecting against infection by diverse microorganisms, including bacteria [1].The key constituents of the innate immune system in defence against invading microorganisms include physical barriers, antimicrobial peptides, complement system factors, antibodies, collectins, and ficolins as freely secreted antimicrobial proteins found in the blood, as well as leukocytes such as polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes [2,3].However, microbial pathogens have evolved mechanisms to evade, suppress, and manipulate innate immune defences.These pathogens often target the initial points of microbial recognition and can influence host cell signalling, production of pro-inflammatory cytokines, and trafficking and secretion of proteins.The balance between immune activation and suppression is intricately regulated, allowing an optimal host response against infection while simultaneously limiting collateral immunemediated damage to host tissues [4].
Adenosine (Ado), a key biomolecule implicated in modulating the host response to infections and tissue damage, is a particularly potent immunosuppressive signal.Ado is produced from ATP, ADP, and AMP, catabolized by 5'-nucleotidases, which are distributed across all life forms and various cellular locations [5,6].Ado not only prevents over-exuberant inflammation to protect the host during acute infections but might also harm the host by dampening the protective antimicrobial response; therefore, the balance between ATP/ ADP/AMP and Ado concentration is important in immune homoeostasis [4,7].Thus, 5'-nucleotidases that convert ATP, ADP, and AMP to Ado are involved in cell-matrix or cell-cell interactions and transmembrane signalling and play important roles in immune and inflammatory responses.5'-Nucleotidases can be classified based on their mechanism of hydrolysis and the type of molecule used as the initial acceptor of the substrate phosphoryl group: an activated water molecule for the type I catalytic mechanism or a nucleophilic amino acid residue for the type II catalytic mechanism [8].Microbial 5'-nucleotidases that utilize the type I catalytic mechanism have been identified and characterized in Gram-positive (Staphylococcus, Streptococcus, and Bacillus species) and Gram-negative (Vibrio, Shewanella, and Legionella species) bacteria.These 5'-nucleotidases show low but significant sequence identity with mammalian ecto-5'nucleotidase CD73 [5,9], indicating their common ancestry and similar structures [10,11].The N-terminal domain of these proteins, which contains a dimetal centre and the catalytic dyad Asp-His in the active site, is characteristic of the calcineurin-like phosphoesterase superfamily (PF00149.28) of phosphatases, while the C-terminal domain is characteristic of the 5'-NT superfamily (PF02872.18)[6].In Gram-positive bacteria, 5'-nucleotidases increase Ado levels, thereby helping these bacteria compromise the host's immune defences and survive in host tissues during infection [12], and in some cases also convert dAMP to dAo to trigger caspase-3-mediated death of immune cells [13][14][15].Streptococcus suis (S. suis) is an important Gram-positive porcine pathogen and zoonotic agent that causes septicaemia, meningitis, arthritis, and many other diseases [16][17][18].Since the first reported cases of human S. suis infection in Denmark in 1968, more than 1600 cases have been reported worldwide [19].Among S. suis 29 serotypes, serotype 2 (SS2) is the most virulent and prevalent one in swine and humans worldwide [20].SS2 causes two large-scale outbreaks that occurred in 1998 and 2005 in China resulting in 229 infections and 52 deaths [21], particularly because of the cases presented with streptococcal toxic shock-like syndrome (STSLS).Bacterial pathogens evade host innate immune defences and maintain a high dose in blood causing bacteraemia and septicaemia.During these processes, the precise role and mechanism of 5'-nucleotidases in the virulence of S. suis remains to be elucidated.
In this study, we identified and characterized four Streptococcal 5'-nucleotidases (Snts) in S. suis that are capable of converting ATP, ADP, and AMP into the immunosuppressive molecule Ado to varying extents.Moreover, it was demonstrated that Ado generation capacity is positively correlated with enhanced bacterial survival in whole blood and PMNs, colonization within the blood and specific organs in vivo, and lethality rate in infected mice.These insights contribute to the expanding knowledge of the multifaceted functions of 5'-nucleotidases, underscoring their significant impact on bacterial pathogenicity.

Bacterial strains and culture conditions
S. suis serotype 2 strain SC-19 (GenBank accession number: CP020863.1)used in this study was isolated from a sick pig during an epidemic outbreak in the Sichuan Province of China in 2005 [22].The S. suis strains were grown in brain heart infusion (BHI; Oxoid, United Kingdom) broth or on BHI agar plates supplemented with 5% newborn bovine serum (Tianhang, China) at 37°C.The bacterial strains and plasmids, primers used in the present study are listed in Table 1 and S1, respectively.
Recombinant plasmids were transformed into E. coli Rosetta (DE3) competent cells (Tsingke, China).The resulting E. coli cells in the mid-log phase were induced by 1 mmol/L isopropyl-β-d-thiogalactoside (IPTG) (Sangon, China) at 16°C for 16 h to express recombinant proteins.His-tagged proteins were purified by Ninitrilotriacetic acid (Ni-NTA) resin (Sigma, USA) affinity chromatography.All the proteins were purified by applying gravity-assisted flow to disposable columns.Purified proteins were quantified using a BCA protein assay kit (Beyotime, Shanghai, China) and subjected to 10% SDS-PAGE.

Construction of gene deletion and site mutant strains
Four markerless single-gene deletion strains retained 6 amino acids at the N-terminal and C-terminal of each gene, and 1 four-gene deletion strain was constructed via a two-step procedure as described previously [23].Take sntA single-gene deletion strain, for example, in the first step, primers sntA-P1/P2, sntA-P5/P6, and sntA-P3/P4 were used to amplify the upstream and downstream arms of sntA, SCIY cassette, respectively.These three resulting DNA fragments were fused into one fragment using overlapping PCR and transformed into S. suis SC-19 by natural transformation to obtain the intermediate strain.In the second step, primers sntA-1-P1/P7, and sntA-P8/P6 were used to amplify the upstream and downstream arms of sntA, respectively.These two resulting fragments were fused and transformed into the intermediate strain by natural transformation to obtain markerless sntA gene deletion strain ΔsntA.The sntB, sntC and sntD single-gene deletion strains ΔsntB, ΔsntC, and ΔsntD were constructed as described above.The four-gene deletion strain was constructed based on ΔsntA through deletion of sntB, sntC and sntD genes one by one.Four single-site mutant strain sntA H209A , sntB H161A , sntC H132A , sntD H82A were constructed also as described above with some modifications.Taken sntA H209A as example, the His-209 residue was transformed to Ala (cac→gca) using primers sntA-H209A-F/sntA-H209A-R in order to generate site-mutagenesis sntA gene.The N-terminal and C-terminal segments of site-mutagenesis sntA gene were amplified by primers sntA-P1/sntA-H209A-R and sntA-H209A-F/sntA-P6, respectively.Subsequently, these two resulting segments were fused into one intact sitemutagenesis sntA gene using overlapping PCR and transformed into the intermediate strain mentioned before by natural transformation to obtain site mutant strain sntA H209A .The single-site mutant strains sntB H161A , sntC H132A , and sntD H82A were constructed through mutation of the corresponding His residue to Ala (cat→gca) as described above.The four-site mutant strain was constructed based on sntA H209A through mutation of other three His residues in sntB, sntC, and sntD one by one.
The sntA, sntB, sntC, and sntD complementary strains CΔsntA, CΔsntB, CΔsntC, and CΔsntD were constructed as the method mentioned before [24].To construct sntA complementary strain, a DNA fragment containing the entire sntA coding sequence and its promoter was amplified by using primers pSET2-sntA -F/R.The amplicon was subsequently cloned into E. coli-S.suis shuttle vector pSET2, resulting in the recombinant plasmid pSET2:sntA.This plasmid was transformed into the makerless ΔsntA strain, and the resulting complementary strain CΔsntA was screened on TSA agar plates supplemented with 100 μg/ml spectinomycin (Spc).The other complementary strains CΔsntB, CΔsntC, and CΔsntD were constructed as described above.All the gene deletion strains and complementary strains were verified using genomic PCR and RT-PCR.

Enzymatic activity assays
Phosphate (Pi) was detected using malachite green reagent following the manufacturer's recommendations (Biomol Green, Enzo Life Sciences, USA).The reaction was carried out at 37°C in TM buffer (50 mM Tris-HCl, 5 mM MnCl 2 , pH7.5) with increasing concentrations of 0-100 μM ATP (Sigma), ADP (Sigma), and AMP (Sigma) and 0.1 μM recombinant SntA, SntB, SntC, SntD, and their related site-mutagenesis enzymes in a total volume of 50 μL for 30 min.After stopping the reaction with 1 ml of Biomol Green reagent, the samples were incubated at room temperature for 20-30 min to allow the development of the green colour.The Pi concentrations were determined by spectrophotometric absorbance measurements at 620 nm using a standard Pi curve.To investigate the effect of pH, temperature, and cofactors on the reaction, the reaction was carried out at different temperature of 22-52°C in 50 mM Tris-HCl adjusted to different pH values (between 6 and 8.5) containing 50 µM AMP, 5 mM different cofactors, and 0.1 μM recombinant SntA, SntB, SntC, or SntD enzymes.The enzyme kinetics were analysed by incubation of increasing amounts of 0-500 µM AMP for Snts (exception for 0-2, 000 µM AMP for SntD) with a fixed concentration of 0.05 μM Snts enzyme at 37°C in a total volume of 50 μL.The reactions were stopped and detected as described above.Michaelis-Menten curve fitting using nonlinear regression was performed using GraphPad Prism 9 software.
The generation of Ado was determined by HPLC using a Prominence UFLC LC20AD (Shimadzu, Japan) with a reverse-phase InertSustain C18 column (Shimadzu, 4.6 mm × 250 mm, 5 µm).Reactions were performed in TM buffer containing 500 µM AMP and 10 µM recombinant Snts in a total volume of 400 µL at 37°C for 30 min.The enzymatic reaction was stopped by the addition of EDTA to a final concentration of 50 mM.The reaction samples (20 µL) were loaded and eluted in a linear solvent gradient with 100% methanol running from 2% to 70% (2%, 2-50%, 50-65%, 65-70%, 70%, 70-2%, and 2% methanol) in 20 mM NH 4 H 2 PO 4 /30 mM K 2 HPO 4 buffer at a flow rate of 1 mL/min.AMP and Ado were detected at an absorbance wavelength of 254 nm.Commercial chromatographic grade AMP and Adenosine (Sigma) were used as standards.

Bacterial enzymatic activity assays
The S. suis strains were grown to the middle-log phase and 50 μL S. suis strains cultures (2 × 10 9 CFU/mL) were collected at 2, 800 g for 5 min.The culture precipitate was resuspended in the TM buffer and mixed with 1 mM AMP.A total volume of 400 μL of each compounds was added to the reaction at 37°C for 30 min.Reactions were stopped and detected using both the malachite green reagent and HPLC.

Whole blood killing assay
Blood-killing assays were performed as described previously [24].Mixtures of 1 × 10 4 CFU S. suis strain cultures and 450 μL fresh mouse blood were incubated at 37°C for 0.5 h.Exogenous adenosine (10 μM; Sigma), 5'-nucleotidase inhibitor (5'-(α, β-methylene) diphosphate) (500 µM; Sigma), and sterile ddH 2 O as a control were added to these mixtures.Live bacteria were counted by plating serially diluted samples on BHI agar.The survival index of live bacteria was subsequently calculated as CFU after incubation /CFU in original inoculum .Data are presented as the mean ± standard deviation (SD) from three separate replicates.The experiments were repeated three times.

Mouse infection assay
To detect the effect of Ado synthesis on the virulence of S. suis, a mouse model was used as described previously [24].Sixty female 5-week-old specific-pathogen -free (SPF) ICR mice were acquired from the Laboratory Animal Centre of Hangzhou Medical College, Hangzhou, Zhejiang Province, China.These mice were randomly divided into six groups (10 mice per group) and intraperitoneally infected with 3 × 10 8 CFU/mouse of S. suis.The survival rate of mice was recorded at 12 h intervals for 7 days.To evaluate the effect of Ado synthesis on survival in the bloodstream and colonization of distant organs in mice, a total of 72 female 5-week-old SPF ICR mice (six mice per group) were intraperitoneally infected with 1 × 10 8 CFU/mouse of S. suis.Bacterial counts in the blood, brain, kidneys, and lungs were collected at 12 and 24 h post infection (hpi).Bacterial colonies in various tissues were analysed on BHI agar plates, as described previously.Flowcharts of animal study were shown in Figs.S1 and S2.All the mice in each group were housed in separate cages (4-5 mice per cage) at 21-26°C and 50-60% humidity.

Identification of adenosine synthase
The N-terminal His-tagged SntA (93 KDa), SntB (79 KDa), SntC (76 KDa), and SntD (55 KDa) were characterized following their expression in E. coli, purified via affinity chromatography, and confirmed by SDS-PAGE analysis (Figure 2a).Enzymatic assays using the malachite green reagent method revealed the differential abilities of these enzymes to produce inorganic phosphate (Pi) from ATP, ADP, and AMP, with notable increases in Pi production from ATP by SntA, SntB, and SntC, an unremarkable increase in SntD (Figure 2b), and significant increases in ADP and AMP across all enzymes, indicating their varying capacities to convert these substrates into adenosine (Figure 2c,d).Further analysis using HPLC confirmed these findings, particularly for AMP hydrolysis (Figure 2e).Gene deletion strains for each nucleotidase (ΔsntA, ΔsntB, ΔsntC, and ΔsntD), a collective deletion strain (Δsnts), and complementary strains for each deletion (CΔsntA, CΔsntB, CΔsntC, and CΔsntD) were constructed and verified using genome-PCR and RT-PCR (Fig. S3).The AMP hydrolysis ability of natural SntA, SntB, SntC, and SntD in S. suis SC-19 was analysed using both malachite green reagent and HPLC.The results showed that the Ado generation capacity was significantly reduced in ΔsntB, ΔsntC, ΔsntD, and strongly decreased in Δsnts, with no significant change observed in the ΔsntA strain (Figure 2f-g).This suggests an additive role of SntB, SntC, and SntD in adenosine synthesis, which is supported by the lack of change in adenosine production among S. suis SC-19 and all complementary strains, indicating that naturally occurring SntB, SntC, and SntD retain their functional capacity for AMP hydrolysis.Consequently, it was demonstrated that four recombinant nucleotidases, along with their natural counterparts excluding SntA, can hydrolyse ATP, ADP, and AMP to various extents, thereby contributing to the production of the immunosuppressive molecule, adenosine.

The pH, temperature, metal cofactors, and kinetics specificity of Snts
The enzymatic activity of recombinant Snt proteins was evaluated under various conditions, revealing distinct preferences for metal cofactors.Specifically, SntA activity was enhanced by Co 2+ , Mn 2+ , Ca 2+ , and Mg 2+ , whereas SntB was stimulated by Mn 2+ , Ca 2+ , Mg 2+ , and SntD by Co 2+ , Mn 2+ , Ca 2+ , and SntC showed no dependency on cations, indicating that Mn 2+ is the necessary metal cation for the enzymatic activity across Snts (Figure 3a).In contrast, all the Snts displayed minimal to no activity in the presence of 5 mM Zn 2+ .The enzymes demonstrated optimal pH values for activity at pH 7.0 for SntA and SntC, and pH 7.5 for SntB and SntD, aligning closely with the pH of blood and extracellular fluids of the body (Figure 3b).The optimal temperature for enzymatic activity was 37°C, which approximates the human body temperature and is slightly below that of pigs (Figure 3c).Kinetic analyses following the Michaelis-Menten model highlighted differences in the initial rate of Pi release with varying concentrations of AMP.The kinetic parameters were determined as Km and Vmax for each enzyme: SntA exhibited a K m of 13.67 μM and    3d).These findings underscore the nuanced catalytic efficiency of Snts towards AMP, with SntC being the most efficient, followed by SntB, SntA, and SntD.
The His-Asp catalytic dyad, which is critical for enzymatic function, is conserved across SntB, SntC, and SntD, with a notable deviation in SntA, where Asn replaces Asp.Metal ion-and phosphate-binding sites were preserved across all Snts, indicating a shared structural framework for their enzymatic activity (Figure 4a).Additionally, other essential residues from the 5'-nucleotidase signatures, GNHEFD, were conserved across these enzymes (Figure 4b).Structural alignment analysis between S. suis 5'-nucleotidase with the mammal CD73 revealed different coverage rates for Snts (SntA: 62%; SntB: 63.9%; SntC: 71.5%; SntD: 98%), with SntD exhibiting the highest coverage.Mutation analysis of SntD, focusing on its catalytic, metal ion binding, phosphate binding, and nucleotide-binding sites, confirmed the critical roles of these residues in the generation of Ado (Figure 4c-d).Moreover, Gly-80, Glu-83, and Phe-84 in the 5'-nucleotidase signatures were first discovered to be important for AMP hydrolysis, but not for the key cores (Figure 4d).Interestingly, mutation analysis of SntA with altered residues did not account for its limited 5'-nucleotidase activity, suggesting that other factors are at play (Figure 4d).These results suggest that the enzymatic mechanism is similar to that of SntB, SntC, SntD, and the known 5'-nucleotidases.Further experiments with mutant recombinant proteins in which histidine residues critical for activity were altered to alanine (SntA H209A , SntB H161A , SntC H132A , and SntD H82A ) showed a complete loss of Ado generation, as confirmed by both the malachite green assay and HPLC (Figure 5a-b).Similar results were observed in single site-mutagenesis strains (sntA H209A , sntB H161A , sntC H132A , sntD H82A ), confirming the essential nature of these histidine residues for the enzymatic activity of Snts (Figure 5c-d).Collectively, these data highlight the crucial role of specific residues in the 5'-nucleotidase activity of Snts and their similarity to known 5'nucleotidases, highlighting the conservation of enzymatic mechanisms across different species.

Ado generation increases the anti-opsonophagocytosis of S. suis
To assess the impact of Ado generation on the antiopsonophagocytosis capabilities of S. suis, whole blood and PMNs killing assays were performed.In a mouse whole-blood killing assay, the survival rates of sntB H161A , sntC H132A and particularly the snts* mutant strains, were significantly lower than those of wild-type S. suis SC-19.No obvious differences in survival were observed between the sntA H209A and sntD H82A mutants and wild-type strain (Figure 6a).The addition of exogenous adenosine to S. suis strains resulted in increased survival across all the site-mutagenesis strains, with the sntB H161A , sntC H132A and snts* strains showing significant increases of 16.75%, 19.03%, and 45.18%, respectively (Figure 6b).The application of a 5'-nucleotidase inhibitor (5'-(α, β-methylene) diphosphate) led to a marked reduction in the survival of SC-19 and the single-mutagenesis strains, whereas the snts* strain showed no significant change (Figure 6c).Similar results were observed in the PMNs killing assay, regardless of the addition of exogenous adenosine or 5'nucleotidase inhibitor (Figure 6d-f).These results demonstrate that Snts collectively contribute to bacterial survival in both whole blood and PMNs, although the roles of SntB and SntC, which exhibit strong enzymatic activity, are more critical than those of SntA and SntD, which exhibit limited activity.Adenosine decreases phagocytic activity in macrophages by suppressing the generation of nitric oxide [28], superoxide [29,30] and pro-inflammatory cytokines [31] through signalling via the A2a receptor.The use of an A2aR 5'-nucleotidases using malachite green reagent (f) and HPLC (g).The data were represented as means ± standard deviations (SD) from three independent experiments.Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparison test (a-d) and unpaired t-test (f), denoted by asterisks (***p<0.001;ns, p>0.05).antagonist to investigate the role of A2aR in antiopsonophagocytosis resulted in reduced survival rates for all strains, except for the snts* mutant strain (Figure 6g), indicating that the Ado generation activity of Snts enhances bacterial survival in PMNs through the Ado-A2aR pathway, without ruling out the involvement of other Ado receptors.In summary, the capacity of Snts to generate adenosine promotes bacterial survival in whole blood and PMNs by engaging the Ado-A2aR pathway, highlighting a direct correlation between adenosine generation and augmentation of bacterial resistance against opsonophagocytosis.

Ado generation increases the S. suis survival and virulence in mouse
Initial observations confirmed that there was no significant difference in the growth rate between the wildtype and mutant strains sntA H209A , sntB H161A , sntC H132A , sntD H82A (Fig. S4).The direct influence of the Ado generation on S. suis virulence was further examined using mouse infection models.Groups of 10 healthy SPF mice were intraperitoneally infected with 3 × 10 8 CFU of various S. suis strains.The outcomes revealed significant differences in mortality rates at 72 hpi, with nine mice infected with SC-19 and sntD H82A , and eight mice infected with sntA H209A succumbing to the infection.In contrast, no or minimal mortality was observed with sntB H161A , sntC H132A , and snts* within 168 hpi, indicating survival rates of 100%, 90%, and 100% for these strains, respectively, compared with 10% and 20% for SC-19 and sntA H209A (Figure 7a).These findings revealed that mutations affecting Ado generation in sntB H161A , sntC H132A , and snts* significantly reduced mortality compared with the parental SC-19 strain, suggesting a critical role for Ado generation in S. suis virulence.sntA H209A and sntD H82A also reduced mortality despite of no significant difference comparing with S. suis SC-19.Further analysis of intraperitoneal infections of groups of seven mice with 1 × 10 8 CFU of S. suis strains, followed by assessments of blood and organ colonization at 12 and 24 hpi.The colonization efficiency of sntB H161A , sntC H132A and snts* exhibited marked reductions in blood, lung, brain, and kidney colonization compared to that of SC-19, particularly for the snts* strain, which displayed the most pronounced attenuation (Figure 7b).No significant differences were observed between sntA H209A and sntD H82A comparing with S. suis SC-19.These findings underscore that the Ado generation capability of SntB and SntC is essential for the virulence, survival in the blood, and organ colonization of S. suis.Additionally, these results suggest a direct positive correlation between the Ado generation capacity and the ability of S. suis to survive and colonize the host, contributing to the lethality rate of infected mice, which highlights the importance of Ado generation in the pathogenicity and host interaction of S. suis.

Discussion
Adenosine (Ado), synthesized by 5'-nucleotidases, plays a dual role in modulating inflammation during acute infections by mitigating excessive immune responses; however, it may also compromise the host's antimicrobial responses.This delicate equilibrium between ATP/ ADP and Ado concentrations is crucial for the maintenance of immune homoeostasis [32].S. suis is an important swine bacterial pathogen that can be transmitted to humans and is the leading cause of severe systemic diseases such as meningitis and septicaemia with sudden death in piglets, and STSLS was first reported in humans in 2005 [21,33].Despite their significance, the roles and mechanisms of 5'-nucleotidases in the virulence of S. suis remain poorly understood.
Our study shows that four cell wall-anchored 5'nucleotidases of S. suis effectively catalyse the conversion of ATP and ADP, demonstrating a correlation with increased susceptibility in the blood and PMNs, and enhanced survival and virulence in vivo (Figure 8).5'-Nucleotidases can be found in a wide variety of species, including mammals, protozoa, plants, fungi, and bacteria, and use AMP and ADP, and in some cases ATP, to produce the immunosuppressive Ado, which dampens pro-inflammatory immune responses [34].Several streptococcal 5'-nucleotidases have been functionally characterized, including S. sanguinis Nt5e [13], S. agalactiae NudP [35], S. pyogenes S5nA [26]    human pathogenic species, and S. iniae S5nAi [36], S. suis Ssads [37], and S. equi subsp.zooepidemicus 5Nuc [15] from animal pathogens that occasionally cause infections in humans [32].The zoonotic S. suis SC-19 harboured four 5'-nucleotidases (Snts), SntA, SntB, SntC, and SntD, which share analogous domains but possess low sequence identity (Figure 1 and Table 2).These enzymes additively process ATP, ADP, and AMP into Ado, with SntB and SntC exhibiting high activity, and SntA and SntD showing limited activity in vivo (Figure 2).Multiple sequence alignment of each Snt with CD73 [25], E. coli 5'-nucleotidase [27] and S5nA [26] has revealed that the substrate-binding pocket, His-Asp catalytic dyad, metal ion binding, and phosphatebinding sites are fully conserved in recombinant SntB, SntC, SntD, and all the reported 5'-nucleotides [32], suggesting that the enzymatic mechanism is similar for 5'-nucleotidases.In addition, the structural alignment other established 5'-nucleotidases.The sites of his-asp catalytic dyad and metal ion binding are marked above using asterisks (*).analysis between CD73 with S. suis 5'-nucleotidases reveals a higher identity of SntB (34.5%),SntC (34.7%) than SntA (24.7%),SntD (27.63%), suggesting that the different enzymatic activity of S. suis 5'-nucleotidases may be attributed to the different structural identity.Besides, at a concentration of 0.1 μM SntA (which nearly releases 0.25 nmol Pi) was found to produce less adenosine than SntD (which nearly releases 1.5 nmol Pi) when both enzymes were exposed to an equal amount of 50 μM AMP in vitro (Figures 2d and 3a-c).This outcome is surprising given that SntA's catalytic efficiency was higher than that of SntD (Figure 3d).The likely explanation for this discrepancy is the significantly lower Vmax of SntA (0.2025 μM/min) compared to SntD (1.546 μM/ min).This suggests that the maximum amount of product that can be produced by SntA is limited in comparison to SntD, even under the same conditions.Consequently, the deletion of sntsD had a detectable effect whereas the deletion of sntsA did not (Figure 2f).It is important to note that the relative expression levels of SntD and SntA in S. suis are currently unknown.Evenly, SntA exhibits no detectable activity in vitro in previous report [38].Another previous study report that His-117 (His-209 in SntA) is important in the catalytic activity of S. enterica [39] and E. coli [40] CpdB which belong to metallophosphatase family, and Tyr-440 and Tyr-544 (Tyr-530, Tyr-633 in SntA) can form a sandwich with the nitrogen base of substrates such as 3'-AMP [39,41].The amino acid sequence identity between SntA with S. enterica CpdB, E. coli CpdB are 45.82% and 46.02%, respectively, indicating that SntA is more likely to belong to the CpdB-like metallophosphatase family better than 5'-nucleotidase family.Despite some differences in substrate specificity, pH range, metal ion requirements, and catalytic efficiency, all characterized 5'-nucleotidases can hydrolyse AMP to immunosuppressive Ado, which dampens proinflammatory immune responses and hydrolyzes dAMP to dAdo, triggering caspase-3-dependent apoptosis in macrophages and preventing phagocytic killing of the bacteria [32].Portions of 5'-nucleotidases, such as NudP [35,37], AdsA [12], S5nAi [42], S5nA [43], and Nt5e [13] have been shown to convert AMP to Ado and relate to survival in host blood and specific tissues and virulence in animal models of infection.However, the effect of 5'-nucleotidases on virulence is attributed to their enzymatic activity or other mechanisms, such as protein-protein interactions.In this study, to emphasize the importance of enzyme activity, sitemutagenesis strains with deletion of AMP hydrolysis capacities to different extents were used to illustrate the direct relationship between Ado generation activity and virulence in vitro and in vivo.This indicates that 5'nucleotidases not only contribute to survival in blood, PMNs, and virulence in mouse models directly, but there is also a positive correlation between their Ado generation activities and survival in blood, PMNs, and mice (Figures 6 and 7), clearly laying out the importance of 5'-nucleotidases in virulence in vitro and in vivo.
In S. suis serotype 2, SsnA plays a key role in neutrophil extracellular trap (NET) degradation, resulting in a significant susceptibility against the antimicrobial effect mediated by NETs, although EndAsuis can also degrade NET [44].Nucleotidases usually work in synergy with nucleases to generate dAdo and have distinct activities [32].S. suis serotype 9 NT (100% identity with SntB) can convert DNA-derived deoxyadenosine monophosphate (dAMP) into 2'deoxyadenosine (dAdo) to trigger caspase-3-dependent death of mouse macrophages.Further observations have provided direct evidence that S. suis NT synthesizes dAdo in mouse blood, which causes monocytopenia in mouse blood in vivo.In addition, the in vivo transcriptome analysis in mouse blood shows that the inhibitory effect of NT on immune responses and neutrophil functions may be mediated through the generation of Ado [45].In conclusion, S. suis serotype 2 can produce Ado to increase survival in the blood and PMNs in vitro and survival and virulence in a mouse model in vivo, and may produce dAdo to cause monocytopenia although the activity of Snts to convert dAMP to dAdo has not been experimentally demonstrated.
In conclusion, this study firmly establishes the additive effects of S. suis 5'-nucleotidases in generating Ado, contributing significantly to the pathogen's ability to evade host immune responses, thereby directly affecting its survival, virulence, and host-pathogen interaction dynamics.This research not only advances our understanding of S. suis virulence factors but also contributes to the broader field of infectious diseases, providing a foundation for the development of innovative treatments against bacterial pathogens.
(a) Survival analysis of 5-week-old female ICR mice following intraperitoneal infection with 3 × 10 8 CFU/mouse of S. suis SC-19 and its sitemutagenesis strains sntAH209A, sntBH161A, sntCH132A and sntDH82A.Each group consisted of 10 mice.Significant difference in survival between different groups was analysed by log rank (mantel-cox) test for trend.(b) Quantification of bacterial loads in blood, brain, lung, and kidney.Six mice per group were intraperitoneally infected with 1 × 10 8 CFU/mouse of S. suis.Results were shown as log 10 of recovered bacterial counts (CFU/mL in blood and CFU/g in organs), excluding abnormal death.Statistical significance was determined by unpaired t-test, denoted by asterisks (***p < 0.001; **0.001).

Figure 3 .
Figure 3. Determining the optimal conditions for S. suis 5'-nucleotidases. from (a) The effect of different metal ions Co 2+ , Cu 2+ , Zn 2+ , Mn 2+ , Ca 2+ , and Mg 2+ on the enzymatic activity.(b) The effect of varying pH levels (ranging from 6 to 8.5) on the enzymatic activity.(c) The effect of temperature changes (22-52 °C) on the enzymatic activity.(d) The Michaelis-Menten kinetics of the enzymes, analysed with increasing AMP concentrations were plotted and fitted using nonlinear regression in GraphPad prism 9 software.The data were represented as means ± standard deviations (SD) from three independent experiments.Statistical significance was determined by unpaired t-test and denoted by asterisks (***p < 0.001; **0.001).

Figure 4 .
Figure 4. Analysis of catalytic sites in S. suis 5'-nucleotidases.(a)Conserved core residues among SntA, SntB, SntC, and SntD were predicted by multiple sequence alignment based on CD73, E. coli 5'nucleotidase, and S5nA.(b) Multiple sequence alignment of two signature sequences critical for 5'-nucleotidase activity across Snts and (c) Purification of recombinant mutant SntD and SntA proteins, verified by SDS-PAGE gel analysis.(d) Evaluation of the hydrolysis activity of the mutant proteins SntD and SntA towards AMP.The data were represented as means ± SD from three independent experiments.Statistical significance was determined by unpaired t-test, denoted by asterisks (***p < 0.001; *0.01).

Figure 6 .
Figure 6.Impact of adenosine and inhibitors on S. suis survival in blood and PMNs.

Figure 7 .
Figure 7. Evaluating the role of 5'-nucleotidases in S. suis pathogenicity using mouse infection models.

Figure 8 .
Figure 8. Schematic representation of 5'-nucleotidase enzymatic activity and its contribution to S. suis virulence.The figure illustrates how cell wall-anchored 5'-nucleotidases in S. suis additively generate the immunosuppressive molecule adenosine (Ado) with varying efficiency, and how this production directly influences the bacterium's ability to survive in the blood and PMNs, as well as its virulence in mouse models (a-d).SntA and SntD, which have a lower efficiency in generating Ado, have been shown to play a minimal role in affecting the bacterium's susceptibility to blood and PMNs and its overall virulence (b).Conversely, the pronounced Ado production capabilities of SntB and SntC are critical; their absence significantly diminishes survival in the blood and PMNs, as well as virulence in mouse models (c), underscoring the vital role of effective Ado generation by these enzymes in S. suis pathogenicity.

Table 1 .
Bacterial strains and plasmids used in this study.